Camptothecin, a plant alkaloid derived from the Chinese Camptotheca acuminata tree, was added to the National Cancer Institute's natural products screening set in 1966. It showed strong anti-neoplastic activity but poor bioavailability and toxic side effects. After thirty years of modifying the camptothecin scaffold, two derivatives emerged and are now approved for clinical use1. Topotecan (Hycamptin®; GlaxoSmithKline) is currently employed to treat solid ovarian, lung and brain tumors1. CPT-11 (also called Irinotecan, and Camptosar®; Pfizer) contains a carbamate-linked dipiperidino moiety that significantly increases bioavailability in mammals1. This dipiperidino group is removed from the CPT-11 prodrug in vivo by carboxylesterase enzymes that hydrolyze the carbamate linkage to produce the drug's active metabolite, SN-382. CPT-11 is currently used to treat solid colon, lung and brain tumors, along with refractory forms of leukemia and lymphoma3.
The sole target of the camptothecins is human topoisomerase I4. This enzyme relieves superhelical tension throughout the genome and is essential for DNA metabolism, including DNA replication, transcription and homologous recombination5. Topoisomerase I breaks one strand in duplex DNA, forming a covalent 3′-phosphotyrosine linkage, and guides the relaxation of DNA supercoils6,7. It then reseals the single-strand DNA break and releases a relaxed duplex DNA molecule. The camptothecins bind to the covalent topoisomerase I-DNA complex and prevent the religation of the broken single DNA strand, effectively trapping the 91 kDa protein on the DNA4. Such immobilized macromolecular adducts act as roadblocks to the progression of DNA replication and transcription complexes, causing double-strand DNA breaks and apoptosis3. Because cancer cells are growing rapidly, the camptothecins impact neoplastic cells more significantly than normal human tissues. Structural studies have established that the camptothecins stack into the duplex DNA, replacing the base pair adjacent to the covalent phosphotyrosine linkage8,9. Religation of the nicked DNA strand is prevented by increasing the distance between the 5′-hydroxyl and the 3′-phosphotyrosine linkage to >11 Å8,9.
CPT-11 efficacy is severely limited by delayed diarrhea that accompanies treatment10. While an early cholinergic syndrome that generates diarrhea within hours can be successfully treated with atropine, the diarrhea that appears ˜2-4 days later is significantly more debilitating and difficult to control11. CPT-11 undergoes a complex cycle of activation and metabolism that directly contributes to drug-induced diarrhea11. CPT-11 administered by intravenous injection can traffic throughout the body, but concentrates in the liver where it is activated to SN-38 by the human liver carboxylesterase hCE1. The SN-38 generated in the liver is conjugated in the liver to yield SN-38 glucuronide (SN-38G)12. SN-38G is excreted from the liver via the bile duct and into the intestines. Once in the intestines, however, SN-38G serves as a substrate for bacterial glucuronidase enzymes in the intestinal flora that remove the glucuronide moiety and produce the active SN-3813. SN-38 in the intestinal lumen produced in this manner contributes to epithelial cell death and the severe diarrhea that limits CPT-11 tolerance and efficacy. This effect has been partially reversed in rats using the relatively weak (IC50=90 μM) β-glucuronidase inhibitor saccharic acid 1,4-lactone14.
While broad-spectrum antibiotics have been used to eliminate enteric bacteria from the gastrointestinal tract prior to CPT-11 treatment15, this approach has several drawbacks. First, intestinal flora play essential roles in carbohydrate metabolism, vitamin production, and the processing of bile acids, sterols and xenobiotics16,17. Thus, the partial or complete removal of gastrointestinal bacteria is non-ideal for patients already challenged by neoplastic growths and chemotherapy. Second, it is well established that the elimination of the symbiotic gastrointestinal flora from even healthy patients significantly increases the chances of infections by pathogenic bacteria, including enterohemorrhagic E. coli and C. difficile18-24. Third, bacterial antibiotic resistance is a human health crisis, and the unnecessary use of antimicrobials is a significant contributor to this problem19. For these reasons, we pursued the targeted inhibition of gastrointestinal bacterial glucuronidases rather than the broad-spectrum elimination of all enteric microflora.
Glucuronidases hydrolyze glucuronic acid sugar moieties in a variety of compounds25. The presence of glucuronidases in a range of bacteria is exploited in commonly-used water purity tests, in which the conversion of 4-methylumbelliferyl glucuronide (4-MUG) to 4-methylumbelliferone (4-MU) by glucuronidases is assayed to detect bacterial contamination26. Whereas relatively weak inhibitors of glucuronidase have been reported27, no potent and/or selective inhibitors of the bacterial enzymes have been presented. Thus, there is a need for selective inhibitors of bacterial glucuronidase with a purpose of reducing the dose-limiting side effect and improving the efficacy of the CPT-11 anticancer drug.